Plasmids of incompatibility group P (IncP) are remarkable for their extraordinary host-range among gram-negative bacteria. While this potential has been exploited to develop genetic systems for a variety of interesting bacterial species, the genetic and molecular basis for the promiscuity of IncP plasmids is not known. The objective of these studies on the IncP plasmid RK2 is to understand how these plasmids control their replication and ensure their stable maintenance in such a wide variety of gram-negative bacteria. It is now clear that control of RK2 replication involves an unusual and complex regulatory network. The key elements are the kor genes (korA, korB, korC, and korE) which were discovered by this laboratory for their control of the curious host-lethal kil determinants (kilA, kilB1, kilB2, and kilC). The kor genes control expression of each of the kil determinants, the recently revealed rac genes located near kilA, the korA-korB operon, and the trfA operon, which encodes initiator functions for replication at the RK2 origin of replication (oriV). While the functions of the kil genes are unknown, their co-regulation with the plasmid replication gene makes them primary candidates for functions involved in plasmid host-range and stable maintenance. With one exception, the regulation involve two or more kor functions in different combinations. Thus, the system has a general significance for understanding multicomponent regulation of gene expression. This proposal describes genetic and molecular experiments to determine the function of the kil genes, beginning with a detailed analysis of the kil operon, the use of phage lambda transducing phages to determine the mechanism of kilA-mediated cell death and to isolate kil-resistant mutants of the host and phage, and construction of mutations in individual kil gene and their transfer into RK2 for studies of replication and host-range. The kor products will be purified and studied with respect to interactions with their target in the DNA and their possible interactions with each other. Genetic studies will be done to test whether RK2 encodes an alternative replication system, to determine the functions of all the trfA gene products, to identify host functions that interact with the trfA initiator, and to test for post- segregational killing of plasmidless cells by RK2. It is anticipated that these studies will lead to an understanding of how RK2 manages stable maintenance in different bacteria.